Many samples (e.g., of chemical, biological or environmental sample) cannot be injected into chromatographic, nuclear magnetic resonance, or other analytical equipment without prior sample pre-cleaning steps to remove interferences such as particulate matter and soluble contaminants. Injecting such samples without pre-cleaning will lead to temporary or permanent system contamination. Thus labs dealing with such samples often spend 50-70% of their overall analysis time preparing samples for injection into these delicate instruments, including time for enriching and/or concentrating fluid samples.
Current approaches to pre-cleaning are typically offline e.g., U.S. Pat. Nos. 6,200,533 and 6,124,012), and generally involve placing samples in solid phase extraction (SPE) cartridges (typically composed of a polymer such as polyethylene.) SPE cartridges typically comprise tubular or syringe-shaped components filled with a separation medium such as silica gel or derivatized silica gel. Inteferences (components suspended in the sample) are extracted after loading upon the separation medium by washing and eluting. SPE may be tailored to capture or pass specific components in the liquid sample. Analyses of biological samples such as plasma and urine using HPLC generally require SPE to remove both particulate matter and soluble contaminants. SPE can also be used to perform a simple fractionation of a liquid based upon differences in the chemical structure of the component parts.
Existing SPE procedures have a number of disadvantages, including requiring a great deal of manual labor to pipetting the sample and solvents through SPE columns or cartridges. Variations in pipetting volume, speed and time contribute to poor process reproducibility. Reproducibility is a cornerstone to validating manufacturing and/or testing processes, such as, for example, for the purposes of conforming to good manufacturing practices (GMPs) and/or good laboratory practices (GLPs.) Furthermore, the size of conventional SPE devices makes analysis of small volume samples difficult.
Fluid flow through or over the separation medium in the conventional SPE columns and cartridges is driven by gravity or by vacuum (such as described in U.S. Pat. Nos. 4,090,850 and 6,491,873) to an outlet at their respective bottoms. Typically, a separate collection tray or container receives the filtrate passing through the cartridge(s). Samples often flow through the SPE material at different speeds, leading to partial or even complete absorption of the analytes. Thus, some SPE cartridges may run dry during elution, whilst others still have a sufficient amount of solvent.
Multi-well filtration apparatus are also well known in the art, and may be used for the assay of biological liquids. Conventional multi-well filtration plates have 96, 384, or 1536 wells for performing multiple assays simultaneously. Each well is adapted to contain a sample fluid, having a relatively small internal volume defined in part by an interior surface extending downwardly from an opening at an upper surface of the multi-well plate to a bottom outlet and collection tray.
After samples have undergone SPE or filtration processing, they may be injected into the chromatographic instruments manually or by means of autosamplers, automated instruments adapted to receive and/or grasp and manipulate sample containers, such as well plates, trays or vials containing the samples to be injected. Through alignment and movement (typically in multiple dimensions) of one or more injector needles with respect to indexed positions of the sample containers, metered samples are withdrawn and injected into chromatographic instruments. The movement of the needle(s) is guided by a robotic controller executing user programming. Autosamplers that operate on stationary, indexed, multi-well trays, such as Series 1100 HPLC Autosamplers manufactured by Agilent Technologies of Palo Alto, Calif., are in wide use. Alternatively functioning autosamplers are also well known in the art, including those that operate upon racks of individual sample vials, and/or those configured for use with rotatable trays. U.S. Pat. Nos. 6,148,680, 5,286,652, 6,468,475 describe some of these types of autosamplers, all of which may be utilized with the present invention.
One online (real-time) process of solid phase micro extraction has been developed that involves disposing polymer-coated fibers within the injector needle of an autosampler. The fiber is pushed into the sample and allowed to absorb analytes. This method has gained acceptance in water analysis, but to date has been met with limited success in analyzing more viscous matrices. Another online approach utilizes a modified CTC-PAL autosampler integrating independent X-Y-Z movable robotic arms. The capital costs of the analytical instrument and the additional robotic manipulator are frequently so high as to negate any productivity gained by the automation.
It would, therefore, be desirable to automate the SPE instrumentation and method in a relatively inexpensive manner. A system that accurately, robustly and reproducibly moves the SPE process into an online, standard analytical workflow, leveraging conventional autosampling equipment, would be of great benefit. A further benefit would accrue to any instrument that enables analysis of biological samples by HPLC (LC/MS) methods.